JPS6219520A - Novel antithrombotic based on glycosaminoglycan, preparationand drug composition - Google Patents
Novel antithrombotic based on glycosaminoglycan, preparationand drug compositionInfo
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- JPS6219520A JPS6219520A JP61162926A JP16292686A JPS6219520A JP S6219520 A JPS6219520 A JP S6219520A JP 61162926 A JP61162926 A JP 61162926A JP 16292686 A JP16292686 A JP 16292686A JP S6219520 A JPS6219520 A JP S6219520A
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- antithrombotic
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Diabetes (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
(57)【要約】本公報は電子出願前の出願データであるた
め要約のデータは記録されません。(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】
本発明は、グリコサきノブリカンの種々の成分からなる
混合物を基にした新しい抗血栓剤、およびその調製法に
関するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antithrombotic agent based on a mixture of various components of Glycosacium nobrican and to a process for its preparation.
本発明は、さらに前述の混合物の薬剤組成物にも関する
ものである。本発明は特に、抗トロンビンｌ （ＡＴＩ
）に高い親和性のあるグリコサミノグリカン抗血栓剤
に関するものである。The invention further relates to pharmaceutical compositions of the aforementioned mixtures. The invention particularly relates to antithrombin I (ATI)
) is a glycosaminoglycan antithrombotic agent that has a high affinity for
ある種のムコ多糖類が血液凝固機能に影響することが知
られている。最もよく知られるムコ多糖び治療に用いら
れている。ヘパリンの抗血栓作用は、抗トロンビンＩに
よる血液凝固機能の阻害を促進嘔せることに基づいてい
る。ヘパリンを血栓症および血栓塞栓症の予防や治療に
用いる際に、出血をおこすというヘパリンの特性が大き
な問題となっている。最も効果的な投与の用量、用法お
よび頻度によシ出血の可能性を幾分は減少させることが
できるが、出血誘発性に対する抗血栓性の比率にはほと
んど影響を及はさない。さらにヘパリンには作用の持続
時間が短かい欠点があシ、十分に予防するためには通常
少なくとも２４時間当−＃）２回の投与が必要である。It is known that certain mucopolysaccharides affect blood coagulation function. It is most commonly used in the treatment of mucopolysaccharide. The antithrombotic effect of heparin is based on promoting the inhibition of blood coagulation by antithrombin I. When heparin is used for the prevention or treatment of thrombosis and thromboembolism, the property of heparin that causes bleeding has become a major problem. The most effective dose, dosage, and frequency of administration can reduce the likelihood of bleeding somewhat, but have little effect on the antithrombotic to hemorrhagic ratio. Furthermore, heparin suffers from a short duration of action, usually requiring two administrations over at least 24 hours for adequate protection.
「改良されたヘパリン」の調製、もしくは改良された性
質を持つへ・クリン類似物質の開発に関して丁でに様々
な試みが成されてきた。例えば、英国特許第２，００２
，４０６号明細書が「改良されたへ／４’ ｌｊン」に
関するもので、この物質は分子量２０００から５０００
ダルトンのオリゴ−へテロ多糖の混合物から成シ、ヘパ
リンの解重合もしくは通常のヘパリン製法の母液中から
単離された後に硫酸化して得られる。このようにして得
られた物質の抗凝固作用に対する抗血栓作用の比率から
ヘパリンに比べて有益性が高いことが示されている。Various attempts have been made to prepare "improved heparin" or to develop heculin analogues with improved properties. For example, British Patent No. 2,002
, 406 relates to "Improved Hen/4'ljn", and this substance has a molecular weight of 2000 to 5000.
It is prepared from a mixture of Dalton's oligo-heteropolysaccharides and is obtained by depolymerizing heparin or by sulfating it after isolation from the mother liquor of a conventional heparin production process. The ratio of anticoagulant to antithrombotic activity of the substance thus obtained indicates that it is more beneficial than heparin.
低分子量の様々の他の「改良されたへ７４リン」（ツー
へバリン）についてはすでに調製でれている。例えば米
国特許明細書３ 、７６６ 、１６７、４，２８１，１
０８．４．３０３，６５１ 、４，３５１，９３８．４
．３９６，７６２．４，４０１，６６２に４．４０１，
７５８．４，４３８．２６１および４，４７４，７７０
参照。A variety of other "improved he74" proteins (two-hebalin) of low molecular weight have been prepared. For example, U.S. Pat. No. 3,766,167,4,281,1
08.4.303,651 , 4,351,938.4
．． 4.401 to 396,762.4,401,662,
758.4,438.261 and 4,474,770
reference.
改良された性質を持つへＡリン類似物質についてはＥＰ
−Ａ−０、０６６、９０８に記載されている。この製品
では抗血栓作用と出血作用が明らかに分離されている。EP for phosphorus analogues with improved properties.
-A-0, 066, 908. The antithrombotic and hemorrhagic effects are clearly separated in this product.
今回発見された新しい抗血栓剤は、ＥＰ−Ａ−０，０６
６，９０８に記載された製品の１分画で、ＡＴＩに対し
高い親和性を示す。The newly discovered antithrombotic agent is EP-A-0,06
A fraction of the product described in US Pat. No. 6,908 shows high affinity for ATI.
ＥＰ−Ａ−０、０６６、９０８に記載されている製品を
以下ＨＥＦと記し、本発明に係る物質を以下）ＩＡ−Ｈ
ＥＦとして示す。The product described in EP-A-0, 066, 908 is hereinafter referred to as HEF, and the substance according to the present invention is hereinafter referred to as) IA-H.
Denoted as EF.
ＨＡ−ＨＥＦはオリゴ糖および（ヘテロ）−多糖（グリ
コサミノグリカン）の混合物から成シ、ヘキソース誘導
体、主にグルクロン酸、イズロｙ酸、グルコサミンおよ
びそれらの硫酸化および／又はアセチル化誘導体がウロ
ン酸誘導体とグルコサミン誘導体が交互に繰シ返えされ
た構造をしている。HA-HEF consists of a mixture of oligosaccharides and (hetero)-polysaccharides (glycosaminoglycans), hexose derivatives, mainly glucuronic acid, iduroic acid, glucosamine and their sulfated and/or acetylated derivatives It has a structure in which acid derivatives and glucosamine derivatives are repeated alternately.
それは白色の非結晶性粉末で、グルノｆ−ミエージョン
クロマトグラフィーによシブキストランに対するその分
子量は５０００Ｄから９０００Ｄ、特に６０００Ｄから
８０００Ｄと決定された。該製品は少量のコンドロイチ
ン硫酸（最高２重量％）およびデルマタン硫酸（ｄｓｒ
ｍａｔａｎｓｕｌｐｈａｔ・）（最高２重量％）を含む
。ガラクトサミン含量はＯから０．１ｍｒｎｏｔ／１１
の間で、通常は０．０７５ ｍｍｏｔ／ｇよシ少ない。It is a white amorphous powder and its molecular weight relative to sibuquitran was determined by gluno f-miagen chromatography to be between 5000D and 9000D, specifically between 6000D and 8000D. The product contains small amounts of chondroitin sulfate (up to 2% by weight) and dermatan sulfate (dsr
(up to 2% by weight). Galactosamine content is from O to 0.1 mrnot/11
between 0.075 mmot/g and usually less than 0.075 mmot/g.
窒素含量は２−３重量％の間で、通常的２．５重量％で
あシ、イオク含量は８〜１２重量％の間で、通常１０−
１０．５重量％であシ、Ｎ−硫酸基の含量は１ｇ当りの
ミリ当量で１．２−１．６であυ、０−硫酸基の含量は
１ｇ当シのミリ当量で１．７−２．１であシ、カルボキ
シル基の含量は１ｇ当シのミリ当量で１．６〜２．０で
′ｊ）！り、Ｎ−アセチル基の含量は１．９＠シのミリ
当量で０．３〜０．６であシ、イズロン酸含量は１．２
〜１．４ ｍｍｏＬ ／ ｇであシ、グルクロン酸含量
は０．３〜０．５ ｍｍｏＬ ／ ７７である。The nitrogen content is between 2-3% by weight, usually 2.5%, and the iodine content is between 8-12% by weight, usually 10-
10.5% by weight, the content of N-sulfate groups is 1.2-1.6 in milliequivalents per gram, and the content of O-sulfate groups is 1.7 in milliequivalents per gram. -2.1, the content of carboxyl groups is 1.6 to 2.0 in milliequivalents per gram ('j)! The content of N-acetyl group is 0.3 to 0.6 in milliequivalents of 1.9@shi, and the content of iduronic acid is 1.2.
~1.4 mmoL/g and glucuronic acid content is 0.3-0.5 mmoL/77.
ＨＡ−ＨＥＦの薬理学的概要について以下のとおシであ
る。The following is a pharmacological overview of HA-HEF.
１）抗凝固活性（ＵＳＰ）は１ｒｎｇ当り１０国際単位
（ＩＵ／ｍ９）以下であり、ヘパリンＵ８Ｐの１分画（
通常５チ以下）にすぎない。1) Anticoagulant activity (USP) is less than 10 international units per rng (IU/m9), and 1 fraction of heparin U8P (
Usually less than 5 inches).
２）抗トロンビン活性はヘノ９リンＵＳＰの５％以下で
ある。2) Antithrombin activity is less than 5% of Heno9rin USP.
３）抗Ｘａ活性は３５０〜４００ＵＡ９（ヘノ９リンＵ
ＳＰの２〜３倍）である。3) Anti-Xa activity is 350-400UA9 (Heno9rin U)
2 to 3 times that of SP).
４）抗血栓活性（Ｕｍ＠ｔａｕモデル）のＩＤ５ｏは静
注で約０．りから０．４＃／ｋｌＦである。4) ID5o of antithrombotic activity (Um@tau model) is approximately 0. 0.4#/kIF.
５）篇ろくべきことに、出血活性に対する抗血栓活性に
関し九有益／危険’４ （ｂｅｎｅｆｉｔ／ｒｌｓｋｒ
暴ｔｌｏ ）は、 ＨＥＦより有益であり、ヘノ譬リン
ＵＳＰに比べ１５〜５０倍有益である・６）半減期はＨ
ＥＦへほぼ等しく、これは少なくともへ一々リンＵＳＰ
Ｋ比べ２倍長いことを意味する。5) Best of all, 9 benefits/dangers regarding antithrombotic activity against hemorrhagic activity.
6) The half-life is more beneficial than HEF and 15-50 times more beneficial than Henolin USP.
Approximately equal to EF, this is at least equal to USP
This means that it is twice as long as K.
さらにＨＡ−ＨＥＦの出血活性は高用量の範囲であって
もやや増加するにすぎず、ＨＥＦよりより低込ものであ
る。これに比べてへｉ４リンＵＳＰの１１１９／に＃の
静注における出血活性は明らかＫきわ立ってお）、高用
量では急激に出血活性が増加する。Moreover, the hemorrhagic activity of HA-HEF only increases slightly even in the high dose range and is much lower than that of HEF. In comparison, the hemorrhagic activity upon intravenous injection of i4lin USP 1119/# is clearly marked (K), and at high doses, the hemorrhagic activity increases rapidly.
この興味深い概要から、 ＨＡ−ＨＥＴが靜脈血橙症お
よび血栓塞栓症の予防や治療にふされしいことが示され
た。本発明物質は何よシも、特に股部の手術（ｈｉｐ
ｏｐ＠ｒａｔｌｏｎ）を受ける又は受は念患者のＤＶＴ
（深部静脈血栓症）症例の予防Ｋｆｆｌしてｈる。低
用量（皮下性）のヘパリンでは該目的に無効であシ、高
用量では出血の可能性が大きいため禁忌である。This interesting overview shows that HA-HET is suitable for the prevention and treatment of phlebotomy and thromboembolism. The substance of the present invention is particularly suitable for groin surgery (hip surgery).
Op@ratlon) is a treatment for DVT in patients with mental illness.
(Deep Vein Thrombosis) Prevention Kffl. Low doses (subcutaneous) of heparin are ineffective for this purpose, and high doses are contraindicated because of the high possibility of bleeding.
ＨＡ−ＨＥＦはたとえ高屈１ｔ（３２００抗ＸａＵ／ｋ
ｌ？／日まで）を用いても、突然変異や毒性があられれ
な−６
ＨＡ−ＨＫＦ ＦｉＨＥＦから種々の方法で得られる。Even if HA-HEF has a high deflection of 1t (3200 anti-XaU/k
l? -6 HA-HKF FiHEF, which does not exhibit mutation or toxicity, can be obtained by various methods.
これらの調製方法に共通な特徴は、ＨＥＦをＡＴＩ［［
と接触させることである。ＡＴＩＩＩと複合したＨＥＦ
の部分をその後非複合部分から分離し、得られた複合物
からＨＡ−ＨＥＦが遊離した後、ＨＡ−ＨＥＦを単離す
る。A common feature of these preparation methods is that HEF is converted to ATI [[
It is to bring them into contact with. HEF combined with ATIII
The portion is then separated from the uncomplexed portion, and after HA-HEF is liberated from the resulting composite, HA-HEF is isolated.
ＡＴＩＩＩはヒト由来のものでも、牛崩漿から単離され
たＡＴＩＩＩのように動物由来のものでも周込られる。ATIII can be derived from humans or from animals such as ATIII isolated from bovine plasma.
ＨＡ−ＨＥＦはパッチ法もしくはカラムを用りて調製さ
れる。HA-HEF is prepared using a patch method or a column.
例えば、ＡＴｆｌｌを）ｆＥＦの緩衝溶液（ｐ）１７．
０〜８．０）中に加え、この混合物をなるぺ〈高温（３
７℃）で時々攪拌−し、形成されたＡＴＩＩＩ複金物を
その後限外濾過ｋ、グ／Ｉ／パーミェーションクロマド
グ９フィーもしくはイオン交換クロマトグラフィーによ
シＨＥＦから分離し、緩衝塩溶液によシ分解した後、進
−ＨＥＦを単離する。For example, ATfl) fEF buffer solution (p) 17.
0 to 8.0) and add this mixture to Narupe (high temperature (3
The ATIII compound formed is then separated from the HEF by ultrafiltration or ion-exchange chromatography, followed by a buffered salt solution. After lysis, the pro-HEF is isolated.
本製法のより有益な方法は、アがロースもしくは適当な
シリカを基礎とした担体材料の如き担体材料に共有結合
したＡＴＩＩを用いることである。それによ）、ＨＥＦ
の複合物は非複合物から容易に分離され、例えばがラス
フィルターでの吸引で戸田される。A more advantageous method of this process is to use ATII covalently bound to a support material such as agarose or a suitable silica-based support material. That's it), HEF
Compounds are easily separated from non-compounds, for example by suction through a lath filter.
担体材料と共有結合したＡＴＩＩ［はカラムの形態でも
使用でき、そこをＨＥＦを含有した溶液を通過させる。ATII [covalently bound to a support material] can also be used in the form of a column, through which the solution containing HEF is passed.
この方法によれば、複合性ＨＥＦおよび非複合性ＨＥＦ
の両方を容易に分離できる。ＨＥＦ溶液をＫしたλＴｌ
ｆｆカラムを通過させる。）ＩＡ−）［ＥＦがカラムに
結合する。カラム洗浄後、１〜３モルの塩化リチウム、
塩化ナトリウム又は塩化カルシウムのような塩溶液によ
りＨＡ−ＨＥＦをカラムから溶出させる。溶出液を透析
又は限外濾過により脱塩し、メタノールで沈殿させるか
又は凍結乾燥によシＨＡ−ＨＥＦを単離する。According to this method, composite HEF and non-composite HEF
Both can be easily separated. λTl of HEF solution
Pass through the ff column. )IA-)[EF binds to column. After column washing, 1 to 3 moles of lithium chloride,
HA-HEF is eluted from the column with a salt solution such as sodium chloride or calcium chloride. The eluate is desalted by dialysis or ultrafiltration and HA-HEF is isolated by precipitation with methanol or by lyophilization.
上述した調製法はそれ自体、へ・母リンの製法として知
られている。これに関連しては、たとえば米国特許明細
書４，１１９，７７４および４，３０１，１５３を参照
。又、たとえばＦＥＢＳ １ａｔｔｓｒ＋ｓ ６６巻
、（１９７６）、９０〜９３ページおよびＢｌｏｃｈｓ
ｍ。The above-mentioned preparation method is known per se as the method for producing hemophosphorus. In this connection, see, for example, US Pat. Nos. 4,119,774 and 4,301,153. Also, for example, FEBS 1attsr+s vol. 66, (1976), pages 90-93 and Blochs
m.
ａｎｄ ＢＩｏｐｈｙｓ、 Ｒｓｃ、 Ｃｏｍｍａ ６
９巻（１９７６）、５７０〜５７７ページ参照。and BIophys, Rsc, Comma 6
9 (1976), pp. 570-577.
へ／母すンを上記したような方法により処理したときは
、ＡＴＩＩＩに対し高い親和性を示すへ・臂リン分画（
ＨＡ−へ・ぐリン）が得られる。ＨＡ−へ・々リンの収
率は約２５〜３５重量％である。ＨＥＦからＩ（Ａ−）
ｔＥＦを生産した場合の収率は約２〜７重量％である。When the hematopoietic phosphorus fraction is treated by the method described above, the hemoglobin fraction, which has a high affinity for ATIII, is produced.
HA-hegulin) is obtained. The yield of HA-helin is about 25-35% by weight. HEF to I(A-)
The yield when producing tEF is about 2-7% by weight.
ＨＡ−へ・々リンの抗凝固活性（ＵＳＰ ）は、へ・母
リンと比べて２〜３倍高−０ＨＡ−ＨＥＦの場合、抗凝
固作用はＨＥＦと比べてほとんど差がない。The anticoagulant activity (USP) of HA-HEF is 2 to 3 times higher than HEF.
氾−ヘノ譬リンの抗Ｘａ活性はヘノ４リンの約２倍であ
る。しかしながら、ＨＡ−ＨＥＦの抗Ｘａ活性は皿Ｆに
比べて３０〜１００倍高い。The anti-Xa activity of Flood-Heno4rin is about twice that of Heno4rin. However, the anti-Xa activity of HA-HEF is 30-100 times higher compared to dish F.
臥−ヘノリンの出血活性に対する抗血栓活性に関する有
益／危険率はへ／ヤリンと比べてほとんど差がなく、い
いかえれば、混−ヘノリンは好ましくない性質であるへ
・Ｉリンの出血誘発性を保持していることになる。しか
しながら、ＨＡ−ＨＥＦの有益／危険率ではＨＥＦに比
べよシ有益であることが知見された。There is little difference in the benefit/hazard ratio regarding the antithrombotic activity of hemo-henolin compared to that of hemo-henoline; in other words, mixed henolin retains the hemorrhagic properties of hemo-henolin, which is an unfavorable property. This means that However, the benefit/risk ratio of HA-HEF was found to be more beneficial than HEF.
この新しいＨＡ−ＨＥＦは、ヘノリンに対し通常なされ
ている方法で、薬剤投与の形態に変換できる。This new HA-HEF can be converted into a form of pharmaceutical administration in the manner normally done for henolin.
例えば注射剤とするため水に溶解し、調剤上許容される
補助剤（防腐剤およびある種の壇類）を添加することも
できる。臨床的応用としては、皮下又は静脈内注射（適
切には間欠的Ｋ）もしくはインヒユージョンとして用り
られる。他の投与方法として、スプレー吸入による肺内
投与、又は軟膏剤やクリームによる経皮投与、又は生薬
による粘膜投与も可能である。For example, for injection, it can be dissolved in water and pharmaceutically acceptable auxiliaries (preservatives and certain ingredients) may be added. In clinical applications, it is used as subcutaneous or intravenous injection (suitably intermittent K) or infusion. Other methods of administration include intrapulmonary administration via spray inhalation, transdermal administration via ointments or creams, or mucosal administration via herbal medicines.
本発明を以下に示した非制限的実施例および試験により
更に説明する。The invention is further illustrated by the non-limiting examples and tests presented below.
（以下余白う
実施例１
十人Ｔｉ１ｌの緩衝溶ｇ（１０ｍｇ／ゴ：０．１Ｍリン
酸塩緩衝液ｐＨ７，８）に１０９／ｌの精製へ・ぐリン
を加え、その後、ｇｐ−人−０，０４３，１５９に記載
されているようにＮ−ヒドロキシサクシニミド基を含む
シリカを基材とする担体材料５０［１＃ｔ−加えた。こ
の混合物を１６時間室温で攪拌し、担体材料を濾過して
取シ出し、０．１ＭＭリン酸塩緩衝液（ＰＨ７，８）で
洗浄し、次いで０．１ Ｍエタノールアミンで洗浄した
。その後、この物質ｆｔＯ〜５℃の温度でエタノールア
ミン（０，１Ｍ ）中で５時間インキュベートし、再び
０．１ Ｍ ＩＪン酸塩緩衝液で洗浄し、続いて３Ｍの
塩化ナトリウムを含む０．１ Ｍリン酸塩緩衝液で処理
し、ヘパリンを除いた。(See the margins below. Example 1 109 g/L of purified Grin was added to 1 L of gp-Ti buffer solution (10 mg/G: 0.1M phosphate buffer pH 7,8), and then 0,043,159 was added to the silica-based support material containing N-hydroxysuccinimide groups. The mixture was stirred for 16 hours at room temperature and the support material The material was filtered off and washed with 0.1 M phosphate buffer (PH 7,8) and then with 0.1 M ethanolamine.The material was then washed with ethanolamine ( 0.1 M) for 5 h and washed again with 0.1 M IJ phosphate buffer, followed by treatment with 0.1 M phosphate buffer containing 3 M sodium chloride to remove heparin. Ta.
実施例２
ＨＡ−ＨＥＦの調製
実施例１で得られたＡＴＩ固定化カラムを０．０２Ｍリ
ン酸塩緩衝液（ｐＨ７，４）で平衡状態とした。Example 2 Preparation of HA-HEF The ATI-immobilized column obtained in Example 1 was equilibrated with 0.02M phosphate buffer (pH 7,4).
ＨＥＦ（２０ＩＩ＃）の緩衝溶液をこのカラム中に通過
させた（カラム材料１！当り５０ｄ）。A buffer solution of HEF (20II#) was passed through the column (50d/1! of column material).
カラムを０．０２酢酸アンモニア緩衝液（ＰＨ８，０）
で１時間洗浄し、それから０．４Ｍ塩化ナトリウム（Ｎ
ａＣｔ）を含むこの緩衝液で再度１時間洗浄した。Column with 0.02 acetate ammonia buffer (PH8,0)
for 1 hour and then 0.4M sodium chloride (N
The cells were washed again for 1 hour with this buffer containing aCt).
その後、カラムｔ−３Ｍの塩化ナトリウムを含む０．０
２Ｍ酢酸アンモニア緩衝液（ｐＨ８，０）で溶出した。Then column t-0.0 containing 3M sodium chloride
Elution was performed with 2M ammonia acetate buffer (pH 8,0).
溶出液を脱塩化し、同時に、中空繊維限外ｒ過装置によ
シ少なくとも１０ダ／１ｎｔの濃度に濃縮した。ＨＡ−
ＨＥＦ ′ｆｔ濃縮溶液から７５％メタノールで沈殿さ
せた。収率は３．６重量％であった。The eluate was desalted and simultaneously concentrated to a concentration of at least 10 Da/1 nt using a hollow fiber ultrafiltration device. HA-
The HEF'ft concentrated solution was precipitated with 75% methanol. The yield was 3.6% by weight.
得られたＨＡ −ＨＥＦの平均分子量は７１００Ｄで。The average molecular weight of the obtained HA-HEF was 7100D.
イオウ含量は１０．３重量％、窒素含量は２．４５重量
％、Ｎ−硫酸基１．３６ミリ当量／Ｉを含み、０−硫酸
基１．８７ミリ当量／ｇを含み、カルゲキシル基１，７
５ミリ当量／１．Ｎ−アセチル基０．４２２ミリ当量／
１１．イズロン酸１．３３ ｍｍｏｔ／Ｉ１．グルクロ
ン酸０．４２ ｍｍｏＡ／Ｉおよびガラクトサミン０、
０７ ｍｍｏｔ／ ９を含む・
実施例３
アガロースゲルマトリックス上にＡＴｌｉを固定化し九
カラムｔ ０．０５ Ｍ ）リス−塩酸（０，１Ｍ塩化
ナトリウム）緩衝ａ（ｐｉ（７，５）で平衡状態とした
。ＨＥＦの同様な緩衝溶液（２ｏＩＩ＃）をこのカラム
中に通過させた（カラム材料１１当り５０ｄ）。Sulfur content is 10.3% by weight, nitrogen content is 2.45% by weight, contains 1.36 meq/I of N-sulfate groups, 1.87 meq/g of O-sulfate groups, 1, 7
5 milliequivalents/1. N-acetyl group 0.422 milliequivalent/
11. Iduronic acid 1.33 mmot/I1. Glucuronic acid 0.42 mmoA/I and galactosamine 0,
Example 3 ATli was immobilized on an agarose gel matrix and 9 columns containing 0.07 mmot/9. A similar buffered solution of HEF (2oII#) was passed through this column (50 d/11 column material).
このカラム全上記緩衝液で洗浄し九。その後、１Ｍ塩化
カルシウムを含む０．０５ Ｍ ） ＩＪス塩酸緩衝液
（ｐＨ７，２）で溶出した。溶出液をデキストラングル
マトリックスカラムを用いたグル濾過によ）脱塩し、得
られた無塩溶液を凍結乾燥し九。Wash this column with all of the above buffers. Thereafter, it was eluted with 0.05 M) IJS hydrochloric acid buffer (pH 7.2) containing 1 M calcium chloride. The eluate was desalted (by gel filtration using a dextrangle matrix column), and the resulting salt-free solution was lyophilized.
得られたＨＡ −ＨＥＦの平均分子量ｔｉ６６００Ｄ、
イオウ含量は１０．１重量％、窒素含量は２．３５重量
％、Ｎ−硫酸基１．３１ミリ当量／１１、〇−硫酸基１
．８２ミリ当量／１１．カル−キシル基１．７８ミリ当
量／１．Ｎ−アセチル基０．４０３ミリ当量／Ｉ、イズ
ロン酸１．２９ｍｍｏｔ／Ｊｌ 、グルクロン酸０、４
７ ｍ ｍｏｔ／ Ｉおよびガラクトサミン０．０４Ｂ
ｍｏｔ／ ｌｌを含む。Average molecular weight of the obtained HA-HEF ti6600D,
Sulfur content is 10.1% by weight, nitrogen content is 2.35% by weight, N-sulfate group 1.31 meq/11, 〇-sulfate group 1
．． 82 milliequivalents/11. Car-xyl group 1.78 meq/1. N-acetyl group 0.403 meq/I, iduronic acid 1.29 mmot/Jl, glucuronic acid 0,4
7 m mot/I and galactosamine 0.04B
Contains mot/ll.
薬理学的試験
実施例２のＨＡ−ＨｇＦ ｆｔＨＥＦ （ＥＰ−Ａ−０
，０６６，９０８の実施例３に従って調製した）、ヘパ
リンおよびＨＡ−へ・臂リンと比較して薬理学的試験を
行なった。HA-HgF ftHEF (EP-A-0
, 066,908), was subjected to pharmacological testing in comparison with heparin and HA-helin.
血液凝固作用
ＨＡ−ＨＥＦ ５ １ ３７０Ｈ
ＥＦ ３ ０．２ ８ヘノ
にリン １７５ １６１ １６
６）ＩＡ−ヘパリン ３７０ ４３７
２８４ＵＳＰ法に従って抗凝固活性を測定した。抗トロ
ンビン活性および抗Ｘ１活性は、精製牛抗トロンビンＩ
ＩＩ、Ｓ−２２３８基質およびＳ −２２２基質の各々
を用いて発色基質法（ｅｈｒｏｍｏｇ＠ｎｉｃ ｓｕｂ
ｓｔｒａｔｓｍｅｔｈｏｄ ）で測定しｆｃ。Blood coagulation effect HA-HEF 5 1 370H
EF 3 0.2 8heno ni rin 175 161 16
6) IA-Heparin 370 437
Anticoagulant activity was measured according to the 284 USP method. Antithrombin activity and anti-X1 activity were determined by purified bovine antithrombin I
II, the chromogenic substrate method (ehromog@nic sub
fc.
抗血栓活性
ラットを用いたＵｍ＊ ｔ ｍ ｕモデル（Ｔｈｒｏｒ
ｎｂｏａｉｓＲｅｓｎａｒａｈ ２７巻、（１９８２）
３５３〜３６３イーゾ）を用いて抗血栓活性を測定した
。Um*tmu model using rats with antithrombotic activity (Thror
nboais Resnarah vol. 27, (1982)
Antithrombotic activity was measured using 353 to 363 Iso).
結果
ＨＡ−）ＩＥＦ １０２
ＨＥＦ ４５
ヘノ９リン ５０ＨＡ−ヘパリン
５５
出血活性
ａ）ラットの皮下出血試験
試験される物質を静注でｊ＋′！、辱し九。１分後、麻
酔したラットの頚動脈を切開して出血させた。３０分間
の出血液を水２０１１１７中に集めた。水中のヘモグロ
ビン濃度を分光光度計で測定し、出血の・臂ラメーター
として用いた（Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｅｈ＠ｍ
１ａｔｒｙ２巻、（１９８３） １７３ページ参照）
。Results HA-) IEF 102 HEF 45 Heno-9 phosphorus 50 HA-Heparin 55 Haemorrhagic activity a) Subcutaneous bleeding test in rats Inject the substance to be tested intravenously j+'! , humiliation nine. One minute later, the carotid artery of the anesthetized rat was incised and bled. The 30 minute bleeding was collected in water 201117. The hemoglobin concentration in water was measured with a spectrophotometer and used as a hemorrhage parameter (Journal of Bioeh@m
1try volume 2, (1983) page 173)
.
結果
ＨＡ−ＨＥＦ １５３５
ＨＥＦ ５１０
ヘパリン １７０
ＨＡ−ヘノぐリン ２００（１） ＴＤ
＝生じ九出血作用の３倍の用量本データはＨＡ−ＨＥ
Ｆの出血傾向がより低いことを示し、これはＨＥＦの２
倍以上有益である。Results HA-HEF 1535 HEF 510 Heparin 170 HA-Henogurin 200 (1) TD
= 3 times the dose of nine hemorrhagic effects This data is based on HA-HE
The bleeding tendency of F is lower than that of HEF.
It is more than twice as beneficial.
ＨＡ−ヘパリンは一般にはヘパリンよシ出血傾向が低い
が、ＨＥＦと比較したＨＡ−ＨＥＦの場合はどはるかに
著しい改良がみられない。Although HA-heparin generally has a lower bleeding tendency than heparin, the improvement is far less significant with HA-HEF compared to HEF.
）ＩＡ−ＨＥＦ（実施例２の物質） 、ＨＥＦおよびヘ
ノ臂リンについて、抗血栓作用の持続時間’５Ｕｍ・ｔ
ａｕモデルを用いて測定した。結果を以下に示す。) IA-HEF (substance of Example 2), the duration of antithrombotic effect '5Um·t for HEF and henolin
It was measured using the au model. The results are shown below.
Claims (10)
）平均分子量５０００から９０００ダルトン、（ｂ）窒
素含量２〜３重量％、（ｃ）イオウ含量８〜１２重量％
、（ｄ）Ｎ−硫酸基の含量１．２〜１．６ミリ当量／ｇ
、（ｅ）Ｏ−硫酸基の含量１．７〜２．１ミリ当量／ｇ
、（ｆ）カルボキシル基の含量１．６〜２．０ミリ当量
／ｇ、（ｇ）Ｎ−アセチル基の含量０．３〜０．６ミリ
当量／ｇ、（ｈ）ガラクトサミン含量０．０〜０．１ミ
リ当量／ｇ、（ｉ）イズロン酸の含量１．２〜１．４ｍ
ｍｏｌ／ｇ、及び（ｊ）グルクロン酸の含量０．３〜０
．５ｍｍｏｌ／ｇから成ることを特徴とする抗血栓剤。(1) Based on a mixture of glycosaminoglycans, (a
) average molecular weight of 5,000 to 9,000 daltons, (b) nitrogen content of 2 to 3% by weight, (c) sulfur content of 8 to 12% by weight.
, (d) N-sulfate group content 1.2 to 1.6 meq/g
, (e) O-sulfate group content 1.7 to 2.1 milliequivalents/g
, (f) Carboxyl group content 1.6-2.0 meq/g, (g) N-acetyl group content 0.3-0.6 meq/g, (h) Galactosamine content 0.0-2.0 meq/g. 0.1 meq/g, (i) content of iduronic acid 1.2-1.4 m
mol/g, and (j) glucuronic acid content 0.3-0
．． An antithrombotic agent characterized in that it consists of 5 mmol/g.
であることを特徴とする特許請求の範囲第１項に記載の
抗血栓剤。(2) The antithrombotic agent according to claim 1, which has an anticoagulant activity (USP) of 10 international units/mg or less.
あることを特徴とする特許請求の範囲第１又は２項に記
載の抗血栓剤。(3) The antithrombotic agent according to claim 1 or 2, wherein the antithrombin activity is 5% or less of heparin USP.
を特徴とする特許請求の範囲第１〜３項のいずれかに記
載の抗血栓剤。(4) The antithrombotic agent according to any one of claims 1 to 3, which has an anti-Xa activity of 350 to 400 U/mg.
０は静注で０．２から０．４ｍｇ／ｋｇであることを特
徴とする特許請求の範囲第１〜４項のいずれかに記載の
抗血栓剤。(5) ID_5_ of antithrombotic activity (Umetsu model)
5. The antithrombotic agent according to any one of claims 1 to 4, wherein 0 is 0.2 to 0.4 mg/kg by intravenous injection.
ンＵＳＰに比べ有益／危険率で１５〜５０倍有益性があ
ることを特徴とする特許請求の範囲第１〜５項のいずれ
かに記載の抗血栓剤。(6) The invention according to any one of claims 1 to 5, characterized in that it is 15 to 50 times more beneficial in terms of benefit/hazard ratio than heparin USP in terms of antithrombotic activity related to hemorrhagic activity. Antithrombotic agents.
とを特徴とする特許請求の範囲第１〜６項のいずれかに
記載の抗血栓剤。(7) The antithrombotic agent according to any one of claims 1 to 6, characterized in that the half-life is at least twice as long as that of heparin USP.
からＡＴIIIを用いたアフイニテイークロマトグラフイ
ーにより得られることを特徴とする特許請求の範囲第１
〜７項のいずれかに記載の抗血栓剤。(8) Claim 1 characterized in that it is obtained from the product described in EP-A-0,066,908 by affinity chromatography using ATIII.
The antithrombotic agent according to any one of items 1 to 7.
を原料として用い、この製品をＡＴIIIと接触させ、Ａ
ＴIIIとの複合物を非複合物から分離し、その複合物を
分解して得られたグリコサミノグリカンの混合物を単離
することを特徴とする特許請求の範囲第１〜８項のいず
れかに記載の抗血栓剤の調整法。(9) Using the product described in EP-A-0,066,908 as a raw material, contacting this product with ATIII and
Any one of claims 1 to 8, characterized in that the complex with TIII is separated from the non-complex, and the mixture of glycosaminoglycans obtained by decomposing the complex is isolated. A method for preparing antithrombotic agents as described in .
剤を含むことを特徴とする薬剤組成物。(10) A pharmaceutical composition comprising one or more antithrombotic agents according to claims 1 to 9.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NL8502009 | 1985-07-12 | ||
| NL8502009 | 1985-07-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6219520A true JPS6219520A (en) | 1987-01-28 |
Family
ID=19846291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61162926A Pending JPS6219520A (en) | 1985-07-12 | 1986-07-10 | Novel antithrombotic based on glycosaminoglycan, preparationand drug composition |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0209924A1 (en) |
| JP (1) | JPS6219520A (en) |
| KR (1) | KR870000930A (en) |
| AU (1) | AU5910686A (en) |
| DK (1) | DK325186A (en) |
| ES (1) | ES8802470A1 (en) |
| FI (1) | FI862897A (en) |
| GR (1) | GR861563B (en) |
| PT (1) | PT82972B (en) |
| ZA (1) | ZA864487B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03167127A (en) * | 1989-10-04 | 1991-07-19 | Akzo Nv | Sulfated glycosaminoglyclonane having antithrombotic action |
| KR100958691B1 (en) * | 2002-01-02 | 2010-05-18 | 딜라포 아베 | Use of sulfated glycosaminoglycans for establishing effective labor in women |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1270823B (en) * | 1993-06-04 | 1997-05-13 | Italfarmaco Spa | HIGH POLYESACCHARIDES WITH HIGH ANTI-THROMBOTIC AND ANTI-COAGULANT ACTIVITY |
| DE19646901A1 (en) * | 1996-11-13 | 1998-05-14 | Helmut Prof Dr Heusinger | Process for the production of degradation products of polymeric glycosaminoglycans by means of ultrasound |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2482611B1 (en) * | 1980-05-14 | 1986-03-07 | Pharmindustrie | NOVEL SULFATED POLYSACCHARIDES, METHODS FOR THEIR PREPARATION AND THEIR USE AS MEDICAMENTS |
| EP0048898B1 (en) * | 1980-09-30 | 1985-04-17 | Bayer Ag | Method for the production of an antithrombin-heparin complex and pharmaceutical compositions containing the complex |
| FR2504928A1 (en) * | 1981-04-29 | 1982-11-05 | Choay Sa | SHORT CHAIN OLIGOSACCHARIDES HAVING BIOLOGICAL PROPERTIES, PREPARATION THEREOF AND APPLICATIONS THEREOF AS MEDICAMENTS |
-
1986
- 1986-06-13 EP EP86201027A patent/EP0209924A1/en not_active Withdrawn
- 1986-06-16 ZA ZA864487A patent/ZA864487B/en unknown
- 1986-06-17 GR GR861563A patent/GR861563B/en unknown
- 1986-06-19 AU AU59106/86A patent/AU5910686A/en not_active Abandoned
- 1986-06-25 ES ES556740A patent/ES8802470A1/en not_active Expired
- 1986-07-08 DK DK325186A patent/DK325186A/en not_active Application Discontinuation
- 1986-07-09 FI FI862897A patent/FI862897A/en not_active Application Discontinuation
- 1986-07-10 JP JP61162926A patent/JPS6219520A/en active Pending
- 1986-07-11 PT PT82972A patent/PT82972B/en unknown
- 1986-07-11 KR KR1019860005594A patent/KR870000930A/en not_active Application Discontinuation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03167127A (en) * | 1989-10-04 | 1991-07-19 | Akzo Nv | Sulfated glycosaminoglyclonane having antithrombotic action |
| KR100958691B1 (en) * | 2002-01-02 | 2010-05-18 | 딜라포 아베 | Use of sulfated glycosaminoglycans for establishing effective labor in women |
| US8207145B2 (en) | 2002-01-02 | 2012-06-26 | Dilafor Ab | Use of sulfated glycosaminoglycans for establishing effective labor in women |
| US8524688B2 (en) | 2002-01-02 | 2013-09-03 | Dilafor Ab | Use of sulfated glycosaminoglycans for establishing effective labor in women |
Also Published As
| Publication number | Publication date |
|---|---|
| ES8802470A1 (en) | 1988-06-16 |
| KR870000930A (en) | 1987-03-10 |
| PT82972B (en) | 1987-12-30 |
| AU5910686A (en) | 1987-01-15 |
| FI862897A (en) | 1987-01-13 |
| EP0209924A1 (en) | 1987-01-28 |
| DK325186D0 (en) | 1986-07-08 |
| ES556740A0 (en) | 1988-06-16 |
| GR861563B (en) | 1986-10-15 |
| FI862897A0 (en) | 1986-07-09 |
| PT82972A (en) | 1986-08-01 |
| ZA864487B (en) | 1987-02-25 |
| DK325186A (en) | 1987-01-13 |
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